Synthetic hepatitis B virus vaccine including both T cell and B cell determinants

ABSTRACT

Chemically synthesized polypeptides include amino acid residue sequences that substantially correspond to the amino acid residue sequences of T cell and B cell determinant portions of a natural, pathogen-related protein, in particular, a hepatitis B virus surface antigen (HBsAg). When administered to a host alone, as polymers or as carrier-bound conjugates, the polypeptides induce the proliferation of thymus-derived cells in hosts primed against hepatitis B virus.

DESCRIPTION

The U.S. Government has rights in this invention pursuant to grantsawarded by the National Institutes of Health.

1. Technical Field

The present invention relates to chemically synthesized polypeptideshaving amino acid residue sequences that substantially correspond to theamino acid residue sequences of T cell and B cell determinant portionsof a natural, pathogen-related protein, in particular, a hepatitis Bvirus surface antigen (HBsAg). When administered to a host alone, aspolymers or as carrier-bound conjugates, the polypeptides induce theproliferation of thymus-derived cells in hosts primed against hepatitisB virus.

2. Background

The present invention relates to the production of novel syntheticantigens based upon information derived from DNA and/or proteinsequences and to the use of those antigens in the production ofvaccines, diagnostic reagents, and the like. More specifically, thisinvention relates to synthetic antigenic polypeptides, which when usedalone, as a polymer or upon coupling to a carrier, immunologicallycorrespond to a T cell and B cell determinant portion of hepatitis Bvirus surface antigen (HBsAg).

Viral hepatitis continues to rank as one of the most importantunconquered diseases of mankind. The general term, viral hepatitis,refers principally to hepatitis A (infectious hepatitis) and tohepatitis B (serum hepatitis), although other known viruses such asyellow fever virus, Epstein-Barr virus and cytomegalovirus can causehepatitis in man. Hepatitis is particularly known for its focal attackon the liver (Greek, hepar), but the disease also influences otherorgans.

In 1965, Blumberg discovered an antigen circulating in the blood ofcertain human beings [J. Am. Med. Assoc., 191, 541 (1965) and Ann. Int.Med., 66, 924 (1967)]. This substance was subsequently found by Princeto be the surface antigen of hepatitis B virus (HBsAg) that is producedin abundance by individuals who are chronically infected with the agent[Proc. Natl. Acad. Sci. (USA), 60, 814 (1968)].

HBsAg has been the subject of extensive immunochemical characterization.Serologic studies show that several strains of the hepatitis B virus(HBV) have one or more determinants in common, which is designated a.Each strain also has two other determinants: either d or y and either wor r. Thus, there are four possible types of the virus: adw, ayw, adrand ayr. The specificity of HBsAg is associated with a singlepolypeptide [Gold et al., J. Immunol., 117, 1404 (1976) and Shih et al.,J. Immunol., 120, 520 (1978)], the entire 226 amino acid sequence ofwhich is established from the nucleotide sequence of the S gene[Tiollais et al., Science, 213, 406 (1981)] of HBV [Valenzuela et al.,Nature (London), 280, 815 (1979); Galibert et al., Nature (London), 281,646 (1979) and Pasek et al., Nature (London), 282, 575 (1979)].

There is an urgent need for a hepatitis B vaccine for groups which areat an increased risk of acquiring this infection. These groups includehealth care and laboratory personnel, and individuals requiring (1)maintenance hemodialysis; (2) repeated blood transfusions or theadministration of blood products; (3) treatment with immunosuppressiveor cytotoxic drugs and (4) treatment for malignant diseases anddisorders associated with depression of the immune response. Inaddition, a vaccine is needed for individuals living in certain tropicalareas where hepatitis B infection is prevalent.

Hepatitis A and B viruses, however, do not multiply significantly incell culture, and there is no current source of laboratory propagatedvirus for vaccine preparation. Indeed, there has been a repeated failureto transmit hepatitis B virus (HBV) serially in tissue or organ cultureswhich has hampered progress towards the development of a conventionalvaccine [Zuckerman, Amer. J. Med. Sci., 270, 205 (1975)].

Classically, a vaccine is manufactured by introducing a killed orattenuated organism into the host along with suitable adjuvants toinitiate the normal immune response to the organism while, desirably,avoiding the pathogenic effects of the organism in the host. Thatapproach suffers from several well known limitations. These vaccines arecomplex and include not only the antigenic determinant of interest butmany related and unrelated deleterious materials, any number of whichmay, in some or all individuals, induce an undesirable reaction in thehost.

For example, vaccines produced in the classical way may includecompeting antigens which are detrimental to the desired immune response,antigens which include unrelated immune responses, nucleic acids fromthe organism or culture, endotoxins and constituents of unknowncomposition and source. These vaccines, generated from complexmaterials, inherently have a relatively high probability of inducingcompeting responses even from the antigen of interest.

In the past, antigens have been obtained by several methods includingderivation from natural materials, coupling of a hapten to a carrier andby recombinant DNA technology. Sela et al. [Proc. Nat. Acad. Sci. (USA),68, 1450 (1971); Science, 166, 1365 (1969); and Adv. Immun., 5, 129(1966)] have also described certain synthetic antigens.

Certain "synthetic" antigens have been prepared by coupling smallmolecules (for example, dinitrophenol) to carriers (such as bovine serumalbumin), thus producing antigens which cause the production of antibodyto the coupled small molecule. The carrier molecule is often necessarybecause the small molecule itself may not be "recognized" by the immunesystem of the animal into which it is injected. This technique has alsobeen employed in isolated instances to prepare antigens by couplingpolypeptide fragments of known proteins to carriers, as described in theabove-referenced Sela et al articles.

While this hapten-carrier technique has served the research communitywell in its investigations of the nature of the immune response, it hasnot been of significant use in the production of antigens which wouldplay a role in diagnostic or therapeutic modalities. One reason for thatdeficiency is that to select and construct a useful antigenicdeterminant from a pathogen (e.g., hepatitis B virus) by this technique,one must determine the entire protein sequence of the pathogen to have areasonable chance of success. Because of the difficulty of this task, ithas rarely, if ever, been done.

Recombinant DNA technology has opened new approaches to vaccinetechnology and has the advantage that the manufacture begins with amonospecific gene; however, much of this advantage is lost in actualproduction of antigen in E. coli, or other organisms. In this procedure,the gene material is introduced into a plasmid which is then introducedinto E. coli which produces the desired protein, along with otherproducts of the metabolism, all in mixture with the nutrient. Thisapproach is complicated by the uncertainty as to whether the desiredprotein will be expressed in the transformed E. coli.

Moreover, even though the desired protein may be produced, there isuncertainty as to whether or not the protein can be harvested or whetherit will be destroyed in the process of E. coli growth. It is well known,for example, that foreign or altered proteins are digested by E. coli.Even if the protein is present in sufficient quantities to be ofinterest, it must still be separated from all of the other products ofthe E. coli metabolism, including such deleterious substances asundesired proteins, endotoxins, nucleic acids, genes and unknown orunpredictable substances.

Finally, even if it were possible (or becomes possible through advanced,though necessarily very expensive, techniques) to separate the desiredprotein from all other products of E. coli metabolism, the vaccine stillcomprises an entire protein which may include undesirable antigenicdeterminants, some of which are known to initiate adverse responses.Indeed, it is known that certain proteins which could otherwise beconsidered as vaccines include an antigenic determinant which inducesserious cross reference or side reactions that prevent the use of thematerial as a vaccine.

It is also possible, using hybridoma technology, to produce antibodiesto viral gene products. Basically, hybridoma technology allows one tobegin with a complex mixture of antigens and to produce monospecificantibodies later in the process. In contrast, the present invention isthe reverse process, in that we start with a relatively high purityantigenic determinant and thus avoid the necessity for purification ofthe desired antigenic product.

Hybridoma antibodies are known to exhibit low avidity and low bindingconstants, and therefore, have limited value. Moreover, in hybridomatechnology, one must rely on the production of the antibody by cellswhich are malignant, with all of the attendant concerns regardingseparation techniques, purity and safety.

Hybridoma production relies upon tissue culture or introduction intomice, with the obvious result that production is costly and there is aninherent variability from lot to lot.

In addition, it is difficult to make hybridomas that secrete antibodiesto molecules which comprise only a small percentage of the complexmixture with which one must start, or which are poorly immunogenic andare overshadowed by stronger, dominant antigens.

Previous studies by Arnon et al., Proc. Nat. Acad. Sci. (USA), 68, 1450(1971), Atassi, Immunochemistry, 12, 423 (1975) and Vyas et al. Science,178, 1300 (1972) have been interpreted by those authors to indicate thatshort linear amino acid sequences are, in general, unlikely to elicitantibodies reactive with the native protein structure. It was thoughtthat for most regions of most molecules, antigenic determinants resultedfrom amino acid residues well separated in the linear sequence but closetogether in the folded protein. The exact three dimensional conformationof the polypeptides used to elicit antibodies was thought to be criticalin most cases, even for those antigens involving amino acids closetogether in a sequence.

For example, Sela thought it necessary to synthesize a rather elaborateloop structure to elicit an anti-lysozyme response. Atassi engineeredmany elaborate molecules, each intended to mimic the tertiary structureof the target protein. And Vyas concluded that the three dimensionalconformation of hepatitis B surface antigen was a critical factor ineliciting antibodies reactive with that native structure.

Sutcliffe et al., Nature, 287, 801 (1980) discovered that antibodies tolinear polypeptides react with native molecules, and recentinvestigations have shown that relatively short chemically synthesizedpolypeptides can elicit antibodies reactive with almost any region of anexposed surface of a protein [Green et al., Cell, 28, 477 (1982)].Moreover, since amino-acid sequences can now be determined rapidly withnucleic acid sequencing technology, synthetic polypeptides can besynthesized to make vaccines of a precision not previously possible.Thus, elaborate biosyntheses are unnecessary, uneconomical and obsolete.

U.S. Pat. No. 4,415,491 to Vyas discloses a series of peptides thatcorrespond to the a determinant of hepatitis B virus surface antigen.Although no data is presented concerning the protection of a host, thepeptides are described as being useful in a hepatitis vaccinepreparation.

Current vaccines for HBV consist of subviral components of the virussurface coat (HBsAg) purified from the plasma of chronicallyHBV-infected donors and inactivated [McAuliffe et al., Rev. Infect.Dis., 2, 470 (1980)]. Clinical trials have demonstrated the safety andefficacy of current HBsAg vaccines but such vaccines are limited insupply and are relatively expensive, particularly for those countrieswith the highest incidence of HBV disease. Chemically synthesizedpolypeptides, therefore, offer considerable advantages in terms of costand safety of HBV vaccination programs.

It is known that antisera to synthetic polypeptides predicted from thenucleotide sequence of various regions of the S gene of HBV react withnative HBsAg by radioimmunoprecipitation [Lerner et al., Proc. Natl.Acad. Sci. (USA), 78, 3403 (1981)] and commercial solid-phaseradioimmunoassays for anti-HBsAg [Gerin et al., in Viral Hepatitis,Szmuness et al (eds.), 49-55 (1982)].

It has been recently determined that a pathogen-related protein can beimmunologically mimicked by the production of a synthetic polypeptidewhose sequence corresponds to that of a determinant domain of thepathogen-related protein. Such findings are reported by Sutcliffe etal., Nature, 287, 801 (1980) and Lerner et al., Proc. Natl. Acad. Sci.(USA), 78, 3403 (1981).

Moreover, Gerin et al., Proc. Natl. Acad. Sci. (USA), 80, 2365 (1983)have recently shown limited protection from hepatitis B virus uponimmunization with carrier bound-synthetic polypeptides having amino acidsequences that correspond to the amino acid sequence of a determinantportion of HBsAg; in particular, residues 110-137.

The construction of a synthetic HBsAg vaccine, however, may require inaddition to synthetic polypeptides corresponding to B cell(antibody-producing) epitopes, synthetic polypeptides corresponding tonon-overlapping T cell determinants.

By way of further background, three cellular components of the immunesystem are B cells (bursa- or bone marrow-derived lymphocytes), T cells(thymus-derived lymphocytes) and macrophages. B cells circulate in theblood and the lymph fluid and are involved in the production ofantibodies. T cells amplify or suppress the response by B cells.

Macrophages, on the other hand, are involved in presenting andconcentrating antigens to B and T cells. Moreover, macrophages secreteseveral biologically active mediators that regulate the type andmagnitude of both T and B cell responses either by enhancing orsuppressing cell division or differentiation. Macrophages arenonspecific and react against any foreign antigen. T and B cell,however, are antigen-specific and react via cell membrane receptors thatare specific for the particular antigen.

In mice, the in vivo antibody production to HBsAg is regulated by atleast 2 immune response (Ir) genes, one in the I-A subregion (Ir-HBs-1)and one in the I-C subregion (Ir-HBs-2) of the murine H-2 complex. It isobserved that immunization with a chemically synthesized peptidecorresponding to the d determinant did not distinguish between high andnon-responder murine strains. Milich et al., J. Immunol., 130, 1401(1983). This suggests that Ir-restriction may occur through T cellrecognition of additional, perhaps nonoverlapping, regions of themolecule.

The linkage between major histocompatibility complex and the regulationof immune responsiveness to HBsAg in mice has been extended to the humanimmune response by the report of an association between HLA-DR phenotypeand nonresponsiveness to a recent trial HBsAg vaccine. Thus, theconstruction of synthetic HBsAg vaccine may require, in addition to Bcell epitopes, a sufficient diversity of T cell determinants toaccommodate the genetic variation in epitope recognition of an outbredhuman population.

The following information would be very valuable in developing asynthetic HBsAg vaccine: (1) whether synthetic peptide fragmentsrepresenting a highly restricted region of the native HBsAg (i.e., about6 amino acids) can induce a T cell proliferative response, which, aswith native HBsAg, is regulated by H-2 linked genes; (2) whether T cellrecognition sites overlap with antibody binding sites; (3) whethermultiple T cell recognition sites exist on HBsAg and if so whether thesite(s) recognized depend on the H-2 genotype of the responding strain;(4) whether the T cell site(s) recognized determine the specificity andquality of the humoral response; and (5) whether human HBsAg-primed Tcells are activated by the same determinants that induce T cellproliferation in mice.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to certain synthetic polypeptides thathave special characteristics and properties, and to products and methodsutilizing those synthetic polypeptides.

Throughout this application, the terms "peptide" and "polypeptide" areused interchangeably. As used herein, the term "synthetic polypeptide"means a chemically built-up, as compared to a biologically built anddegraded, chain of amino acid residues that is free of naturallyoccurring proteins and fragments thereof. Such synthetic polypeptidescan induce the production of anti-polypeptide antibodies in a host.

A synthetic polypeptide in accordance with this invention has an aminoacid residue sequence that is shorter than that of hepatitis B virussurface antigen but includes an amino acid residue sequence thatcorresponds immunologically to that of at least one determinant portionof hepatitis B virus surface antigen (HBsAg).

The polypeptide, when used alone, as a polymer (synthetic multimer) orbound to a carrier such as keyhole limpet hemocyanin (KLH) or the likeas a conjugate and introduced in an effective amount as a vaccine in aphysiologically tolerable diluent such as water, saline and/or anadjuvant into a host animal, can induce the production of antibodies andthe proliferation of thymus-derived cells in the host.

The vaccine is prepared by providing one or more of the followingpolypeptides, a polymer thereof or a carrier-bound conjugate thereof anddissolving or dispersing an effective amount of the polypeptide in aphysiologically tolerable diluent.

Preferred sequences of synthetic polypeptides, for use in a vaccine,comprise amino acid residue sequences (or a portion thereof) of B celldeterminant portions of HBsAg (also referred to herein as Bcell-stimulating and priming portions) taken from left to right and inthe direction from the amino-terminus to the carboxy-terminus including:

    ______________________________________                                        (110)(120)                                                                    Phe(Ile)ProGlySerSer(Thr)ThrThrSerThrGlyProCys                                (130)                                                                         Arg(Lys)ThrCysMet(Thr)ThrThr(Pro)AlaGlnGly                                    (137)                                                                         Thr(Asn)SerMetTyr(Phe)ProSerCys;                                              (125)(130)                                                                    Met(Thr)ThrThr(Pro)AlaGlnGlyThr(Asn)SerMet                                    (137)                                                                         Tyr(Phe)ProSerCys; and                                                        (107)(110)                                                                    CysProLeuPhe(Ile)ProGlySerSer(Thr)ThrThrSerThr                                (120)                                                                         GlyProCysArg(Lys)ThrCysMet(Thr)ThrThr(Pro)Ala                                 (130)(137)                                                                    GlnGlyThr(Asn)SerMetTyr(Phe)ProSerCys                                         ______________________________________                                    

GlnGlyThr(Asn)SerMetTyr(Phe)ProSerCys wherein each amino acid residue inparentheses is an alternative to the immediately preceding amino acidresidue, and the numerals in parentheses above particular amino acidresidues in the above sequences identify positions of the particularamino acid residue relative to the amino-terminus of the hepatitis Bvirus surface protein. Such polypeptides induce the production ofantibodies that can immunoreact with hepatitis B virus and protect ahost from infection.

Preferred sequences of synthetic polypeptides, for use in a vaccine,also include an amino acid residue sequence (or a portion thereof) of aT cell determinant portion of HBsAg that induces T cells to proliferate(also referred to herein as a T cell-proliferating portion) taken fromleft to right and in the direction from the amino-terminus to thecarboxy-terminus as follows:

    ______________________________________                                        (140)(150)(154)                                                               ThrLysProSerAspGlyAsnCysThrCysIleProIleProSer                                 ______________________________________                                    

wherein the numbers in parentheses above particular amino acid residuesin the above sequence identify positions of the particular amino acidresidue relative to the amino-terminus of the hepatitis B virus surfaceprotein.

In addition, vaccine according to the present invention, againstinfection by hepatitis B virus can include an effective amount of asynthetic polypeptide having an amino acid residue sequence thatimmunologically corresponds substantially to an amino acid residuesequence of a natural pathogen-related protein encoded by a hepatitis Bvirus from about positions 107 to 154, 110 to 154 or 125 to 154 from theamino-terminus thereof, and a physiologically tolerable diluent. Thevaccine, when introduced into a host, is capable of inducing theproduction of antibodies and the proliferation of thymus-derived cellsin the host. Such antibodies can immunoreact with hepatitis B virus andthe vaccine can protect the host from hepatitis B viral infection.

For example, the synthetic polypeptide can include the sequence of aminoacid residues taken from left to right and in the direction fromamino-terminus to carboxy-terminus, and represented by the formula:

Phe(Ile)ProGlySerSer(Thr)ThrThrSerThrGlyProCysArg(Lys)ThrCysMet(Thr)ThrThr(Pro)AlaGlnGlyThr(Asn)SerMetTyr(Phe)ProSerCysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIleProSer

wherein each amino acid residue in parentheses is an alternative to theimmediately preceding amino acid residue.

That sequence corresponds to positions 110 to 154 as shown in FIGS. 1and 2. The amino acid residue sequences that correspond to positions 107to 154 and to positions 125 to 154 are also shown in FIGS. 1 and 2.

Each of the above synthetic polypeptides can be used in a monomeric formalone or conjugated to a carrier molecule such as KLH or tetanus toxoid.The synthetic polypeptides can also be used in a multimeric form.

When utilized in multimermic form, each polypeptide is one of aplurality of repeating units of a multimer. In one embodiment, themultimer contains at least two of the polypeptides bonded togetherhead-to-tail through an amide bond formed between the amine group of theamino-terminus of one polypeptide and the carboxyl group of thecarboxy-terminus of the second polypeptide. In another multimericembodiment, the polypeptide is one of a plurality of repeating units ofa polymer whose polypeptide repeating units are bonded together byinterpolypeptide cystine disulfide bonds formed between the Cys residuesof the polypeptide repeating units.

In another embodiment, the present invention includes a diagnosticsystem for determining the presence of cell-mediated immuneresponsiveness to HBsAg and the presence of a hepatitis B virus antigenin a host comprising a synthetic polypeptide as described above that hasan amino acid residue sequence that corresponds to the amino acidsequence of a T cell determinant of HBsAg. The polypeptide, whenadministered to a host intradermally in an effective amount and inphysiologically tolerable diluent, is capable of inducing theproliferation of thymus-derived cells in the host. The proliferation isindicated by erythema (redness) and induration (hardening of the skin)at the site of intradermal administration.

Methods are also disclosed for inducing the proliferation ofthymus-derived cells in a host previously immunized to hepatitis B virusand for determining the presence of a hepatitis B virus antigen in ahost. The methods include the steps of providing a T-cell proliferatingpolypeptide as discussed herein and administering intradermally aneffective amount of the polypeptide to the host in a physiologicallytolerable diluent according to the latter method, the proliferation ofthymus-derived cells and the presence of a hepatitis B virus antigen inthe host is indicated by erythema and induration at the site ofintradermal administration.

The present invention provides several advantages and benefits. Oneadvantage of the present invention is that use of a syntheticpolypeptide obviates the need for the presence of its correspondingintact protein. The polypeptide itself provides a vaccine sufficient toprotect the host from disease. Consequently, impurities such as cellulardebris and toxins that are associated with the production of usableamounts of viral proteins from bacteria are absent from the product ofthis invention.

Moreover, a synthetic hepatitis B virus vaccine having both B cell and Tcell determinants obviates the need to select a carrier appropriate foruse in humans to stimulate the proliferation of thymus-derived cells inthe recipient.

Another benefit of the present invention is that antibodies in antiseraraised to the synthetic polypeptide immunoreact with and can be used todetect the presence of antigenic proteins and polypeptides associatedwith hepatitis B virus.

Still further advantages and benefits of the present invention willbecome apparent to those skilled in the art from the detaileddescription, Examples and claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which constitute a portion of this disclosure:

FIG. 1 illustrates the 226 amino acid sequence of the HBsAg/ayw proteinas translated by Pasek et al., Nature, 282, 575 (1979) from the nucleicacid sequence. Regions of the protein selected for synthesis accordingto the present invention are indicated by bold underlining. Residuesthat are not the same in the three published nucleotide sequencedeterminations are lightly underlined [Pasek et al., Id.; Valenzuela etal., Nature, 280, 815-819 (1979); and Galibert et al., Nature, 281,646-650 (1979)]. The following single letter and three letter codes (SeeFIG. 2) correspond to the indicated amino acids--A, Ala (L-Alanine); C,Cys (L-Cysteine); D, Asp (L-Aspartic acid); E, Glu (L-Glutamic acid); F,Phe (L-Phenylalanine); G, Gly (Glycine); H, His (L-Histidine); I, Ile(L-Isoleucine); K, Lys (L-Lysine); L, Leu (L-Leucine); M, Met(L-Methionine); N, Asn (L-Asparagine); P, Pro (L-Proline); Q, Gln(L-Glutamine); R, Arg (L-Arginine); S, Ser (L-Serine); T, Thr(L-Threonine); V, Val (L-Valine); W, Trp (L-Tryptophan); and Y, Tyr(L-Tyrosine).

FIG. 2 illustrates the amino acid sequences of polypeptides designated1, 5, 5a, 6, 49, 49a, 72, 71, 72a and 73 using the conventional threeletter code for each amino acid. These sequences are read from left toright and in the direction from the amino-terminus to thecarboxy-terminus of the polypeptide. Polypeptides 1, 5, 5a and 6correspond to residues 48-81, 38-52, 47-52 and 95-109, respectively, ofHBsAg. Polypeptides 49 and 72 correspond to residues 110-137 of HBsAg(peptide 73 corresponds to residues 107-137) as predicted from the Sgene nucleotide sequence of HBV DNA from an ayw donor (polypeptide 49)[Galibert et al., Nature (London), 281, 646-650 (1979)]and an adw donor(polypeptide 72 and 73) [Valenzuela et al., Nature (London), 280,815-819 (1979)]. The underlined residues in polypeptides 72 and 73indicate positions of amino acid variability between those sequences andthat of polypeptide 49. Polypeptides 49a and 72a consist of theC-terminal 12 amino acids of polypeptides 49 and 72, respectively(residues 125-137). Polypeptide 71 corresponds to residues 140 to 154 ofHBsAg.

FIG. 3 illustrates the mouse C₃ H.Q strain T cell proliferativeresponses in popliteal lymph node cells primed by HBsAg (ad or aysubtype) induced in vitro by: native HBsAg; P25 (the 1-226 residuesubunit of HBsAg); the following tryptic fragments of P25: P25-1(residues 1-122) and P25-2 (residues 123-226), and P73, P72, P49, P6,P5, P5a and P2 (residues 140-148). As used herein, the letter "P" beforea number means "peptide" or "polypeptide". Proliferation was determinedby incorporation of tritiated thymidine (³ HTdR) into cellular DNA, andwas expressed as a percent response elicited by the immunogen. Theimmunogen HBsAg/ad elicited a proliferation which produced 20,477 countsper minute (cpm) and the immunogen HBsAg/ay elicited a proliferationwhich produced 33,000 cpm.

FIG. 4 illustrates the mouse B10.A strain T cell proliferative responsesinduced by: native HBsAg, P25 (the 1-226 residue); the following trypticfragments of P25: P25-1 (residues 1-122) and P25-2 (residues 123-226);and P73, P72, P49, P6, P5, P5a and P2. Proliferative response doses andmeans of measurement were the same as in FIG. 3. The proliferativeresponses elicited by immunogens HBsAg/ad and HBsAg/ay were 8724 cpm and11,444 cpm, respectively. Details of the assays of FIGS. 3 and 4 areprovided in Sections IV and V.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

Synthetic polypeptides having amino acid residue sequences thatsubstantially correspond to the amino acid sequences of the d (P72) andy (P49) determinants of HBsAg have been synthesized by Lerner et al.,Proc. Natl. Acad. Sci. (USA), 78, 3403 (1981). These polypeptidespossess the antigenic specificity of the native determinants asdemonstrated by their ability to bind anti-native HBsAg antibodies. Inaddition, it has been demonstrated that immunization with P49 conjugatedto keyhole limpet hemocyanin (KLH) induces a high-titered anti-yresponse in a murine inbred responder strain. Milich et al., J.Immunol., 130, 1401 (1983).

However, immunization with free (unconjugated) P49 induces little or noanti-y production. Similarly, free P72 induces a very minimal anti-dresponse. Indeed, the reduced immunogenicity of unconjugated (relativeto conjugated) synthetic peptide analogues of HBsAg has been encounteredby numerous investigators.

Therefore, protein carrier molecules such as KLH and tetanus toxoid havebeen used as a means of providing nonspecific T cell helper function forthese synthetic determinants.

In order to construct a synthetic HBsAg vaccine possessing both T celland B cell determinants, it is first necessary to identify the T celland B cell determinants of HBsAg.

It is known that the murine immune response to HBsAg is regulated byH-2-linked Ir genes, and that this regulation is expressed at the T celllevel. Nonresponder haplotypes are characterized by a defect in T-helpercell function, whereas HBsAg-specific B cell repertoirs are intact. Inaddition to the reduced immunogenicity of free, unconjugated syntheticpeptide analogues of HBsAg, immunization with P72 (residues 110-137) orP49 (residues 110-137) did not distinguish between high responder andnonresponder strains.

These results indicate that P72 and P49 represent B cell epitopes of thenative structure, but lack the appropriate T cell determinants.

Thus, immunization with these B cell epitopes alone does not generatethe necessary Ir-restricted, T cell helper function.

A number of HBsAg synthetic peptides were screened as described hereinin an HBsAg-specific T cell proliferative assay in order to identify Tcell determinants. Mice were immunized in vivo with native HBsAg/ad orHBsAg/ay, and popliteal lymph node (PLN) cells were harvested andchallenged in vitro with either native HBsAg or a series of syntheticpolypeptides.

Several polypeptides were identified that stimulate HBsAg-primed PLNcells to proliferate in vitro. In particular, polypeptides P5 (residues38-52), P5a (residues 47-52), P6 (residues 95-109) and P71 (residues140-154) of FIGS. 1 and 2 stimulate T cell proliferation of murine PLNcells primed in vivo with HBsAg of the ad or ay subtype.

Moreover, at least polypeptide P1 (residues 48-81) and polypeptide P5induce T cell proliferation in human peripheral blood lymphocytes (PBL).

It should be noted that P1, P5, P5a, P6 and P71 do not induce theproduction of antibodies cross-reactive with native HBsAg nor do theybind native anti-HBs antibodies. Conversely, P72 and P49 do not induce(or at best induce only minimal) T cell proliferation, yet bind anti-HBsof the appropriate specificity, and provide some protection againsthepatitis B disease.

These results indicate the existence of distinct loci for T cell and Bcell determinants on the same HBsAg polypeptide. Use of a synthetic Tcell determinant with a B cell determinant, preferably a synthetic Bcell determinant, according to the present invention provides a potentsynthetic antigen.

Previous genetic analysis of the immune responses to HBsAg in H-2congenic, recombinant murine strains predicts the existence of a"carrier-determinant" on HBsAg, since a dominant influence on the immuneresponse to all HBsAg determinants maps to a single Ir gene locus.Polypeptides 5, 5a and 6 correspond to such a carrier-determinant on thenative molecule. These polypeptides function as intrinsic carriers andprovide functional T cell help for any and all synthetic B cell epitopesto which they are coupled.

Thus, one aspect of the present invention is directed to vaccines thatcontain as an active ingredient an effective amount of a Tcell-proliferating polypeptide described herein, e.g. at least one ofpolypeptides 1, 5, 5a and 6. Such a vaccine may be introduced into ahost animal (or a human) after that animal has been immunized with(primed to) a HBsAg B cell activator such as the complete HBsAg moleculeor polypeptides such as those denominated 49, 49a, 72 and 72a. Morepreferably, the T cell-proliferating polypeptide of this invention isadministered to the host animal along with a priming, B cell-stimulatingimmunogen such as polypeptides 49, 49a, 72 and 72a.

The more preferred T cell-proliferating and B cell-stimulating andpriming polypeptides may be introduced into the host as separateentities of one vaccine wherein each is linked to its own carrier or asa homopolymer of active polypeptide repeating units. More preferably,both types of polypeptide are linked to a single carrier and therebyconstitute a single active entity in the vaccine. A synthetic HBsAgvaccine containing co-polymerized polypeptide repeating units with aminoacid sequences that substantially correspond to amino acid sequences ofT cell and also B cell determinants of the native molecule is clearly astill more preferred approach rather than attempting to selectappropriate protein carrier molecule for immunization into humansubjects.

Moreover, enhancement of the immunogenicity of synthetic polypeptidesrelated to HBsAg is a fundamental aspect in the development of asynthetic HBsAg vaccine. The highly immunogenic synthetic HBsAg vaccinedescribed herein has desirable medical as well as economic advantages ascompared to the current human plasma-derived vaccines.

II. Discussion

The data from this study demonstrate that limited regions of thehepatitis B surface antigen (HBsAg) molecule; in particular, residues 48to 81 (which correspond to synthetic peptide P1), residues 38 to 52(which correspond to synthetic peptide P5), residues 95 to 109 (whichcorrespond to synthetic peptide P6) and residues 47 to 52 (whichcorrespond to synthetic peptide P5a) and residues 140 to 154 (whichcorrespond to synthetic peptide 71) are sites that are preferentiallyrecognized by HBsAg-primed T cells.

Although synthetic peptides P1, P5, P5a, P6 and P71 induce T cellproliferative responses, these peptides do not correspondingly induce orbind antibodies that recognize the native molecule. This illustrates thedisparity in determinant specificity that can exist between B and Tcells in response to complex protein antigens.

Such disparity has been observed in a variety of antigenic systems asdescribed in the following references: Senyk et al., J. Exp. Med., 133,1294 (1971); Thomas et al., J. Immunol., 126, 1095 (1981); Berkower etal., Proc. Natl. Acad. Sci. (USA), 79, 4723 (1982); Kipps et al., J.Immunol., 124, 1344 (1980). In contrast, other investigators havedemonstrated similar T and B cell receptor specificities for antigens asdescribed in the following references: Twining et al., Mol. Immunol.,18, 447 (1981); Rajewsky et al., Eur. J. Immunol., 4, 111 (1974); Beckeret al., Eur. J. Immunol., 5, 262 (1975). Any assumption, however, that Tand B cell recognition sites never or always overlap is therefore overlysimplistic.

With reference to HBsAg, C₃ H.Q (H-2^(q)), or simply "C₃ H.Q", andB10.T(6R)(H-2^(q)) or "B10.T(6R)", murine strains preferentiallyrecognize the amino-terminal fragment of HBsAg [in particular, residues1-122 of the P25 HBsAg polypeptide subunit (P25-1)] and the constituentpeptides P5, P5a and P6. Murine strains C₃ H.Q and B10.T(6R) arereferred to herein as "responder strains" or "high responder strains"based on the degree of the proliferative response.

The proliferative response of B10.A (H-2^(a)), or "B10.A", murine strainT cells, on the other hand, is directed almost exclusively to thecarboxy-terminal fragment of HBsAg [specifically, residues 123-226 ofP25 (P25-2)] and to the P72 synthetic peptide, which also serve asantibody binding sites on HBsAg. Murine strain B10.A is referred to asan "intermediate responder strain" because the proliferative response isless than that of C₃ H.Q or B10.T(6R).

Murine strain SJL (H-2^(S)) or "SJL" has been found to confernonresponsiveness to immunization with HBsAg and, thus, is referred toas a "nonresponder strain".

Therefore, multiple T cell recognition sites appear to exist on HBsAgand the selective activation of T proliferating cells is dependent onthe murine major histocompatibility complex (H-2) haplotype of theresponding strain. A similar preferential selection of T cell epitopesin a hapten-carrier system controlled by I-region genes in the murineH-2 complex has been reported in Seman et al., J. Immunol., 129, 2082(1982).

The humoral anti-HBsAg response is regulated by at least two immuneresponse (Ir) genes. One of the genes is in the I-A subregion (Ir-Hbs-1)and the other is in the I-C subregion (Ir-HBs-2) of the murine H-2complex. The Ir-Hbs-1 regulates the response to all HBsAg determinants;whereas, the influence of the Ir-HBs-2 is subtype-specific. (For ageneral description of Ir genes and subregions see Bach, Genetic Controlof Immune Responses in Immunology, ch. 24, pages 677-703 (John Wiley &Sons, New York 1982) which description is incorporated herein byreference).

In the strains used herein, a positive T cell proliferative response tothe amino-terminal fragment of the HBsAg P25 polypeptide subunit P25-1and to synthetic peptides P5a or P6 indicated an enhanced anti-HBsantibody production to all HBsAg determinants. In contrast, the T cellproliferative pattern of the B10.A murine strain corresponds to reducedprimary anti-HBs antibody production which is limited to subtypespecificity.

A site or sites on the amino-terminal fragment of synthetic peptides P5,P5a and P6 serves as a T cell "carrier-determinant" recognized by Thelper cells capable of providing functional help to B cell clonesspecific for the a, d and y epitopes and restricted by the I-Asubregion. In the absence of recognition of the "carrier-determinant,"the influence of subtype-specific helper or suppressor T cellsrestricted by the I-C subregion is observed. Since the B10.A strainproduces a minimal secondary anti-a antibody response, subtype-specificT cells may also provide help to B cell clones specific for theconformational a-epitope.

These observations have important implications in terms of thedevelopment of a synthetic HBsAg vaccine; especially in view of thepossibility that human HBsAg-primed T cells may recognize the sameepitopes as murine T cells.

In particular, the linkage between the major histocompatability complexand the regulation of immune responsiveness to HBsAg in mice has beenextended to the human immune respone. Walker et al., Proc. Amer. Assoc.Blood Banks, 4 (1981) have reported an association between a particularphenotype at the DR gene locus of the human major histocompatibilitycomplex (HLA-DR) and nonresponsiveness to a recent trial HBsAg vaccine.

Thus, the construction of a synthetic HBsAg vaccine preferably includes,in addition to B cell determinants, a sufficient diversity of T celldeterminants to accommodate the genetic variation in epitope recognitionof an outbred human population.

IV. Results

A. Identification of Murine B Cell Epitopes

The polypeptide sequences of hepatitis B surface antigen (HBsAg) thatinduce the production of and bind to murine anti-HBsAg antibodies wereidentified.

A number of polypeptide sequences of HBsAg group a subtype yw(HBsAg/ayw) were selected for synthetic polypeptide synthesis. Thesepolypeptides are denominated P1, P2, P3, P4, P5, P5a, P6, P49, P49a,P72, P72a and P73 and are illustrated in FIG. 1.

The peptides were chemically synthesized by solid-phase methods asdescribed herein in Section VI and as described in greater detail inMerrifield et al., J. Am. Chem. Soc., 85, 2149 (1963) and Houghten etal., Int. J. Peptide Protein Research, 16, 311 (1980). Anti-polypeptideantibodies specific for each of the synthetic peptides were producedwhen the synthetic polypeptides were coupled to KLH and introduced intorabbits as a vaccine that also included water and an adjuvant.

Pooled purified preparations of HBsAg group a subtype d (HBsAg/ad) andHBsAg group a subtype y (HBsAg/ay) were obtained from Dr. Robert Louie(Cutter Laboratories, Berkeley, Calif.). The antibodies to the syntheticpeptides were analyzed for reactivity to HBsAg/ad and HBsAg/ay by ahemagglutination assay (HA) as described herein. The ability of thesolid-phase polypeptides to bind murine anti-native HBsAg antibodies ofd or y specificity was also determined as described below.

Polypeptides P73 (residues 107-137), P72 (residues 110-137) and P72a(residues 125-137) induced the production of antibodies that werecross-reactive with native HBsAg of the ad subtype. Polypeptides P49(residues 110-137) and P49a (125-137), on the other hand, induced theproduction of antibodies that were cross-reactive with native HBsAgprimarily of the ay subtype. (See Table 1).

                  TABLE 1                                                         ______________________________________                                        Identification of B Cell Epitopes On                                          Synthetic Peptide Analogues of HBsAg                                          Anti-peptide reactivity                                                                          Anti-Native HBs Reactivity                                 With Native HBsAg  With Solid-Phase Peptides.sup.2                            HA Titer           RIA Titer                                                  Peptide.sup.1                                                                        HBsAg/ad  HBsAg/ay  Anti-HBs/d.sup.3                                                                       Anti-HBS/y                                ______________________________________                                        P73    1:1280    1:40      1:512    1:32                                      P72    1:160     0         1:1024   0                                         P72a   1:160     0         ND.sup.4 ND                                        P49    1:80.sup.5                                                                              1:160     1:32     1:128                                     P49a   0         1:160     0        1:64                                      P6     0         0         0        0                                         P5     0         0         1:8      0                                         P5a    0         0         0        0                                         P4     0         0         1:4      0                                         P3     0         0         1:16     1:8                                       P2     0         0         0        0                                         P1     0         0         1:16     0                                         ______________________________________                                         .sup.1 Antipeptide antisera were produced in rabbits; and all peptides        were conjugated to keyhole limpet hemocyanin (KLH) with the exception of      P73, P72 and P1. Antipeptide antisera prepared in mice [Milich et al., J.     Immunol., 130, 1401 (1983)]  and chimpanzees [Gerin et al., Proc. Natl.       Acad. Sci. (USA), 80, 2365 (1983)] demonstrate the same specificities for     native HBsAg.                                                                 .sup.2 Peptides (5 micrograms per well) were adsorbed to polystyrene          microtiter plates.                                                            .sup.3 AntiHBs/d and y were produced by immunizing B10.S(9R) mice with        HBsAg/ad or HBsAg/ay, respectively. This H2 recombinant strain produces       only a subtypespecific antibody response.                                     .sup.4 ND = Not determined.                                                   .sup.5 Not specific for the common a determinant.                        

Polypeptides P72 and P72a correspond in amino acid residue positions topolypeptides P49 and P49a, but contain the amino acid substitutionsshown in FIGS. 1 and 2. Although anti-P49 antibodies reacted with bothsubtypes of HBsAg, HA inhibition analysis demonstrated thecross-reactivity was not directed to the common "a"-determinant, butrather to a determinant present on the native HBsAg/ad, P49 and P72 butnot on native HBsAg/ay.

In addition, the ability of the above series of synthetic polypeptidesto bind murine anti-native HBsAg antibodies of d or y specificity wasexamined. As shown in Table 1, P72 bound anti-HBs/d but did not bind toanti-HBs/y; whereas P49 bound anti-HBs/y and anti-HBs/d to some extent(presumably through a cross-reactive determinant not related to thecommon a determinant). The fact that P49a induced the productionanti-polypeptide antibody that reacted only with the ay subtype andbound anti-HBs/y but not anti-HBs/d demonstrates the y-specificity ofthis polypeptide. P73, which is identical to P72 with the addition ofamino-terminal amino acids, demonstrated primarily d-specificity;however, in immunogenicity studies a small cross-reactive component wasobserved. It is interesting to note that HA inhibition analysissuggested this component was specific for a determinant common to bothsubtypes (i.e., anti-a). The remainder of the synthetic polypeptidesused in this determination induced no anti-peptide antibodycross-reactive with HBsAg in non-denaturing conditions and did not bindor bound to a minimal extent anti-native HBsAg antibodies.

These results are in general agreement with those reported by Gerin etal., Proc. Natl. Acad. Sci. (USA), 80, 2365 (1983) and confirm thelocalization of the d and y subtype-specific antibody binding siteswithin synthetic polypeptides P72a and P49a, respectively. Thesesynthetic polypeptides correspond to residues 125-137 of the amino acidsequence of HBsAg, and although P49a differs from P72a at four residues,Peterson et al., J. Biol. Chem., 257, 10414 (1982) have suggested thatamino acid substitutions at residues 131 and 134 confersubtype-specificity.

The minor and low-titered reactivity of anti-native HBsAg with a numberof the other synthetic polypeptides may result from the complexity ofthe antisera that most likely contain specificities directed to HBsAgdegradation products. In support of this position, antisera topolypeptides P3, P4 and P6 do not react with native HBsAg, butnonetheless do bind denatured HBsAg [Lerner et al., Proc. Natl. Acad.Sci. (USA), 78, 3403 (1981)]. The "a-like" activity of P73 may beexplained by the addition of a cysteine residues to P72, since a cyclicform of a synthetic peptide corresponding to residues 122 through 137produced by introduction of an intrachain disulphide bond has beenreported to contain a conformation-dependent a epitope [Ionescu-Matlo etal., J. Immunol., 130, 1947 (1983)].

B. Identification of Murine T Cell Epitopes

The polypeptide sequences of HBsAg that are recognized by native HBsAgprimed mouse T cells were identified.

Native HBsAg/adw was purified from the plasma of a single chroniccarrier as described in Peterson et al., J. Biol. Chem., 256, 6975(1981). P25, a polypeptide subunit of HBsAg, and two tryptic fragmentsof P25 designated P25-1 (residues 1-122) and P25-2 (residues 123-226)were prepared from the same HBsAg/adw positive donor by preparativepolyacrylamide gel electrophoresis as also described in Peterson et al.,supra. Synthetic peptides P73, P72, P49, P6, P5, P5a, P2 and P1 (seeFIG. 1) were synthesized according to the methods described herein.These polypeptides and synthetic peptides were lyophilized, resuspendedin culture media and were sterilized by gamma radiation (5000 rads).

Culture media used was original Click's Media [as described in Click etal., Cell Immunol., 3, 264 (1972)] that was modified by the addition often millimolar HEPES [4(-2-hydroxethyl)-1-piperazinethane-sulfonic acid]and ten micrograms per milliliter gentamycin and by the substitution of0.5 percent syngenic normal mouse serum for fetal calf serum. The P25,P25-1 and P25-2 were not completely soluble in the culture media. Thepolypeptides and synthetic peptides suspended in culture media arereferred to herein as antigens and were cultured with harvestedpopliteal lymph node (PLN) cells as described below.

C₃ H.Q(H-2^(q)) is an inbred murine strain that produces early (10 days)IgG antibodies to both the common a subtype and the d/ determinantsfollowing immunization with HBsAg as described in Milich et al., J.Immunol., 130, 1395 (1983). Groups of five C₃ H.Q mice were immunized inthe rear footpads with an emulsion of complete Freund's adjuvant (CFA)and sixteen micrograms of a pooled purified preparation of HBsAg/ad orHBsAg/ay (obtained as described earlier). Twelve days later popliteallymph node (PLN) cells were harvested and cultured in vitro (2.5×10⁶cells per milliliter) with the antigens that were produced as describedabove.

The antigens were tested in culture over a dose range, however, theproliferative responses illustrated in FIG. 3 correspond to thefollowing in vitro doses: native HBsAg (1.0 micrograms per milliliter);P25, P25-1, P25-2 (10 micrograms per milliliter); and synthetic peptidesP73, P72, P49, P6, P5, P5a and P2 (100 micrograms per milliliter).

HBsAg specific proliferative response of PLN cells harvested up to 13days post-immunization was due to proliferating T cells as described inMilich et al., J. Immunol., 130, 1401 (1983). Consequently,unfractionated PLN cells were used in the determinations.

HBsAg specificity was demonstrated by the absence of antigen-inducedproliferation in CFA-primed PLN T cells. Proliferation was determined byincorporation of tritiated thymidine (³ HTdR) into DNA and was expressedas a percent of the response elicited by the antigen. Assays wererepeated on at least three separate occasions.

The T cell proliferative response is expressed as a percentage of thatinduced by the synthetic polypeptide. HBsAg/ad-primed PLN T cells fromC₃ H.Q mice responded in vitro to native HBsAg/adw almost as well as tothe synthetic polypeptides used, and substantially less to nativeHBsAg/ay, which represents proliferation directed towards commongroup-specific determinant(s) (FIG. 3a).

Polypeptide P25 induced T cell proliferation to the same extent as thenative HBsAg-adw from which it was prepared. Although micrograms permilliliter of P25 was required as compared to 1.0 microgram permilliliter of native antigen, the P25 preparation was not completelysoluble in the culture media, and the effective dose may have beensubstantially less than 10 micrograms per milliliter. This is ofinterest since P25 binds anti-HBs antibody approximately 300-fold lessefficiently than the native antigen.

P25-1 induced a better proliferative response than P25-2 (67 percent vs.45 percent) at 10 micrograms per milliliter, and significantly greaterproliferation at 2.5 micrograms per milliliter (53 percent vs. 13percent). The superior proliferative response induced by P25-1 ascompared to P25-2 in this strain was confirmed by the fact thatsynthetic polypeptides P73 and P72, constituents of P25-2, inducedminimal proliferative responses; whereas P6 (residues 95-109), P5(38-52) and P5a (47-52), constituents of P25-1, induced significantproliferation in HBsAg/ad-primed mice (FIG. 3a). Induction of T cellproliferation by synthetic polypeptides required a 100-fold excess on aweight basis and a 10⁴ -fold excess on a molar basis as compared tonative HBsAg.

It should be emphasized that P6, P5 and P5a do not induce the productionof antibodies cross-reactive with native HBsAg nor do they bind nativeanti-HBs, and conversely, P73 and P72 induce only minimal T cellproliferation in C₃ H.Q mice, yet induce and bind anti-HBs/d (See Table1). These results indicate the existence of distinct T cell and B celldeterminants on the same HBsAg polypeptide. Polypeptide P5a, althoughonly 6 amino acids in length, induced a greater degree of T cellproliferation than did polypeptide P5. This may be because P5a isderived from an extremely hydrophilic region of the amino-terminalfragment (P25-1) of HBsAg and is considerably more soluble in salinethan is polypeptide P5. As previously discussed, the other largehydrophilic portion of the polypeptide corresponds to the antibodybinding regions primarily located on the carboxy-terminal fragment(P25-2).

To determine subtype-specificity of the T cell responses, C₃ H.Q micewere also primed in vivo with HBsAg of the ay subtype. The proliferativeresponses to native HBsAg of the ad subtype and to the adw-derived P25and tryptic fragments P25-1 and P25-2 were reduced as compared toHBsAg/ad-primed mice. However, the responses to synthetic polypeptidesP6, P5 and P5a were virtually equivalent to the responses induced afterHBsAg/ad priming (FIG. 3b). Polypeptides P73 and P72 were notstimulatory for HBsAg/ay-primed T cells, and P49 did not induce aproliferative response after priming with either subtype of HBsAg.

These results indicate that P73 and P72 represent subtype-specificdeterminants at the T cell and the B cell level. P6, P5 and P5a, on theother hand, represent common T cell recognition sites present on bothsubtypes. This is consistent with the amino acid sequence, since the P6and P5a regions are invariable in the HBsAg sequences determined todate, whereas, the P72 region is variable and amino acid substitutionsin this region dictate subtype-specificity. Gerin et al., Proc. Natl.Acad. Sci. (USA), 80, 2365 (1983) and Lerner et al., Proc. Natl. Acad.Sci. (USA), 78, 3403 (1981). The HBsAg-specificity of these responseswas demonstrated by the absence of proliferation in CFA-primed PLN Tcells in response to HBsAg and its related fragments.

Polypeptide 71

The ability of polypeptide P71 (residues 140-154) to stimulate or inducethe proliferation of thymus-derived cells in hosts primed against HBsAgwas also investigated. Murine strain C₃ H.Q was immunized with HBsAgsubtype ad and HBsAg subtype ay. Mice of a nonresponder murine strain(SJL) were inoculated with HBsAg subtype ad. (See Table 2).

                                      TABLE 2                                     __________________________________________________________________________    Tritiated Thymidine [.sup.3 HTdR] Incorporation (Δ cpm).sup.1           Murine                                                                             Primed                                                                              polypeptide 71 (micrograms/milliliter)                             Strain                                                                             T cells                                                                             100                                                                              50 25   12  3   0.07                                                                             0.03                                         __________________________________________________________________________    C.sub.3 H.Q                                                                        HBsAg/ad                                                                            -- -- 20,299                                                                             20,331                                                                            15,007                                                                            6,832                                                                            3,179                                        C.sub.3 H.Q                                                                        HBsAg/ay                                                                            8,130                                                                            9,694                                                                            11,490                                                                             --  --  -- --                                           SJL  HBsAg/ad                                                                            0  0  0    0   0   0  0                                            __________________________________________________________________________     .sup.1 The change in counts per minute (cpm) after correction for             background readings from the control media.                              

Immunizations were performed as described in Milich et al., J. Immunol.,130, 1395 (1983). Groups of five C₃ H.Q mice were immunized in the rearfootpads with an emulsion of complete Freund's adjuvant and sixteenmicrograms of a pooled purified preparation of HBsAg/ad or HBsAG/ay(obtained as described earlier). Twelve days later popliteal lymph node(PLN) cells were harvested and cultured in vitro (2.5×10⁶ cells permilliliter) in the previously described modified Click's media and inthe presence of polypeptide P71.

As indicated in Table 2, P71 was used in culture over a dose range of0.03 to 100 micrograms P71 per milliliter of solution. HBsAg specificitywas demonstrated by the absence of antigen-induced proliferation in theCFA-primed PLN T cells. Proliferation was determined by theincorporation of tritiated thymidine (³ HTdR) into DNA as describedhereinafter in greater detail.

Note that polypeptide P71 has no measurable effect on nonresponder SJLHBsAg-primed T cells. The data presented in Table 2, however, indicatethat P71 can act as a T cell determinant on the carboxy-terminal regionof the native HBsAg molecule. This is consistent with the ability ofP25-2 (residues 123-266) to induce T cell proliferation in each of theresponder strains studied.

C. Determination of the Role of H-2 Restriction in Mouse T CellRecognition Sites

B10.A is an inbred murine strain which only produces an anti-HBsAg-d/subtype specific response after primary immunization as described inMilich et al., J. Exp. Med., 159, 41 (1984) which is incorporatredherein by reference. Groups of 5 B10.A mice were primed in vivo witheither HBsAg/ad or HBsAg/ay. See FIG. 4. The immunization protocol,culture conditions and preparation, concentration of in vitro antigens,and T cell proliferative testing are the same as described above insection B.

Since the high responder C₃ H.Q strain preferentially recognizes P25-1,P6 and P5 at the T cell level and produces high titered anti-HBs/a andanti-HBs/d or y after primary immunization, it was of interest toexamine T cell responses to these antigens in an intermediate responderstrain which only produces subtype-specific anti-HBs/d or y afterprimary immunization. The B10.A strain respresnts such a strain.

B10.A PLN T cells primed with HBsAg/ad responded to native HBsAg/ad butnot at all to native HBsAg/ay (FIG. 3a). Although P25 was stimulatoryfor B10.A HBsAg/ad-primed PLN T cells, the responses to the trypticfragments P25-1 and P25-2 indicated a preferential response to P25-2rather than P25-1 in contrast to the C₃ H.Q strain. Correspondingly,polypeptides P73 and P72 induced significant proliferation, whereaspolypeptides P6, P5 and P5a were virtually non-stimulatory for B10.A PLNT cells primed with native HBsAg/ad or HBs/ay (FIG. 3b).

B10.A mice primed with HBsAg/ay demonstrated only minimal T cellproliferative response in HBsAg/ay-primed mice (FIG. 3a).

These results demonstrate that B10.A HBsAg-primed T cells preferentiallyrecognize the subtype-specific regions of the polypeptide (P25-2 andP72) rather than the group-specific regions as in the case of C₃ H.Qmurine strain. The ability of P72 to stimulate a d-specificproliferative response and induce and bind anti-native HBs/d clearlyindicates that region 110-137 is recognized by both T cells and B cellsin B10.A mice.

To determine the relevance of the above findings to peptideimmunogenicity and in vivo anti-HBs antibody production, C₃ H.Q andB10.A mice were immunized with P72, an analogue of the d determinant,and serum anti-peptide and anti-HBs titers were measured temporally.

As shown in Table 3, following primary immunization with native HBsAg/adthe C₃ H.Q strain produced subtype and group-specific anti-HBs. TheB10.A strain produced only anti-HBs/d, and to a lesser degree than theC₃ H.Q strain. However, following tertiary immunization with P72 theB10.A strain produced a 32-fold greater anti-P72 response and a 20-foldhigher anti HBs/d response as compared to the C₃ H.Q strain (Table 3).

                  TABLE 3                                                         ______________________________________                                        Strain-Dependent In vivo Antibody Production                                  Following Immunization With Synthetic Peptide P72                                         Serum Anti-Polypeptide P72 and                                                Anti-HBs Titers (RIA).sup.2                                       Strain                                                                              Immunogen.sup.1                                                                           Anti-P72 Anti-HBs/ad                                                                            Anti-HBs/ay                               ______________________________________                                        C.sub.3 H.Q                                                                         HBsAg/ad (1°)                                                                      --       1:2,560  1:320                                           P72 (1°)                                                                           0        0        0                                               P72 (2°)                                                                           1:640    1:8      0                                               P72 (3°)                                                                           1:640    1:8      0                                         B10.A HBsAg/ad (1°)                                                                      --       1:320    0                                               P72 (1°)                                                                           1:160    0        0                                               P72 (2°)                                                                           1:2,560  1:20     0                                               P72 (3°)                                                                           1:20,480 1:160    0                                         ______________________________________                                         .sup.1 Groups of 6 mice were immunized with 4.0 micrograms of native          HBsAg/ad or 100 micrograms of peptide P72 in CFA intraperitoneally.           Peptide recipient mice were given identical secondary (2°) tertiar     (3°) immunizations at 2week and 4week intervals, respectively.         .sup.2 Pooled serum antibody titers were measured by solidphase               radioimmunoassay (RIA) and expressed as the highest serum dilution to         yield twice the counts of the preimmunization sera.                      

It should be noted that C₃ H.Q mice immunized with P72 conjugated to acarrier protein produced vigorous anti-P72 and anti-HBs/d responses.Reduced immunogenicity of P72 in the C₃ H.Q strain is consistent withthe inability of P72 to induce a T cell proliferative response inHBsAg/ad-primed C₃ H.Q mice. These results illustrate that the highresponder status of the C₃ H.Q strain is not mediated through T cellrecognition of the subtype-specific d determinant as represented by P72.In contrast, the B10.A strain, which demonstrates P72-induced T cellproliferation following HBsAg/ad immunization, was capable of respondingto P72 immunization with the production of significant concentrations ofanti-P72 and anti-HBs/d antibodies. Therefore, the immunogenicity ofsynthetic peptide analogues of HBsAg is dependent on the requirement forboth T cell and B cell determinants; and the recognition of the T celldeterminant is dictated by the H-2 genotype of the responding murinestrain.

D. Determination of Synthetic Peptides That Elicit HBsAg-Specific T CellProliferation in Mice and are Recognized by Human Vaccine RecipientHBsAg Primed T Cells

Peripheral blood lymphocytes from two human HBsAg/ad vaccine recipients(Haptavax, Merck & Co., Rahway, NJ) designated "DM" and "PW" and anunimmunized volunteer designated "CL" were compared for T cellresponsiveness to native HBsAg/ad, native HBsAg/ay, and a series ofsynthetic peptide analogues of HBsAg.

As shown in Table 4, peripheral blood lymphocytes from one HBsAg vaccinerecipient (DM) responded to native HBsAg of both subtypes and topolypeptides P72 and P5. However, the responses elicited by nativeHBsAg/ad and by polypeptide P72 were significantly greater than thoseelicited by native HBsAg/ay and by polypeptide P5 in terms ofstimulation index and dose response.

In contrast, PBL from (PW) responded equally well to both native HBsAgsubtypes, and correspondingly polypeptides P1 (residues 48-81) and P5induced proliferative responses, whereas polypeptide 72 did not. Theother synthetic peptides tested were not stimulatory nor did any of theantigens stimulate PBL obtained from the nonimmunized control (CL).

Similar to the findings of the murine model, at least two patterns of Tcell specificity were observed in human responses. One pattern ischaracteristic of T cell recognition of distinct determinants (P1 andP5), which do not induce the production of or bind to anti-HBsantibodies. The other pattern involves recognition by T cells of asubtype-specific region that may overlap with B cell determinants.

                                      TABLE 4                                     __________________________________________________________________________    PBL.sup.1 Proliferative Responses of                                          Human HBsAg Vaccine Recipients Challenged                                     In Vitro With Synthetic Peptide Analogues And                                 Native HBsAg                                                                          Anti-HBs status PBL Proliferative Responses (.sup.3 H--TdR; CPM                               and SI).sup.2                                         HBsAg/adw                                                                             (HA Titer)      In Vitro     Antigens.sup.3                           Vaccine HBsAg/ad                                                                            HBsAg/ay                                                                            Media                                                                             HBsAg/ad                                                                             HBsAg/ay                                                                            P72 P49                                                                              P6  P5  P3 P2 P1                  __________________________________________________________________________    (DM) +  1:1600                                                                              1:800 3100                                                                              31,794 20,656                                                                              45,776                                                                            3376                                                                             5583                                                                              11,648                                                                            4442                                                                             5320                                                                             3500                                        (10.3) (6.7) (14.8)                                                                            (1.1)                                                                            (1.8)                                                                             (3.8)                                                                             (1.4)                                                                            (1.7)                                                                            (1.1)               (PW) +  1:8,192                                                                             1:1024                                                                              5000                                                                              14,665 16,958                                                                              6325                                                                              6330                                                                             4784                                                                              10,500                                                                            5683                                                                             ND 22,102                                      (3.0)  (3.4) (1.3)                                                                             (1.3)                                                                            (0.9)                                                                             (2.1)                                                                             (1.1) (4.4)               (CL) -  0     0     2475                                                                              2426   3638  3069                                                                              2723                                                                             2277                                                                              3316                                                                              ND.sup.4                                                                         ND 2000                                        (1.0)  (1.5) (1.2)                                                                             (1.1)                                                                            (0.9)                                                                             (1.3)     (0.8)               __________________________________________________________________________     .sup.1 PBL = Peripheral blood lymphocytes.                                    .sup.2 Human proliferative responses were measured using peripheral blood     lymphoyctes (PBL) and culture conditions modified from the murine assay a     described in LerouxRoels et al., (in press). The .sup.3 H--TdR                incorporation is expressed as counts per minute (CPM) and stimulation         index (SI). A stimulation index is the ratio of the stimulation induced b     the test antigen (measured as counts per minute) to the stimulation           induced by the control media (also measured as counts per minute).            .sup.3 Antigens were used over a wide dose range; proliferative responses     to 1.0 microgram per milliliter of native HBsAg and 100.0 micrograms per      milliliter of peptide analogues are shown. Underscored responses were         greater than two times the media control through at least fourfold            dilutions.                                                                    .sup.4 ND = Not Determined.                                              

V. Materials and Methods

A. Materials

The C₃ HQ, B10.A and SJL inbred murine strains and New Zealand whiterabbits were obtained from the Research Institute of Scripps Clinic, LaJolla, Calif. The B10.T(6R) strain was provided by Dr. Hugh McDevitt(Stanford University, Palo Alto, Calif.). Female mice between 6 and 12weeks of age at the initiation of the studies were used in all studies.

Pooled preparations of HBsAg/ad and HBsAg/ay were provided by Dr. RobertLouie (Cutter Laboratories, Berkeley, Calif.). These preparations werepurified by Cutter Laboratories from human plasma by a combination ofstandard procedures including ultracentrifugation, ammonium sulfateprecipitation, pepsin digestion and gel chromatography. The HBsAgpreparations were free of contaminating human serum proteins as testedby Ouchterlony analysis and immunoelectrophoresis versus goat anti-humanserum [Milich et al., J. Immunol., 129, 320 (1982) which is incorporatedherein by reference].

Native HBsAg/adw was purified from the plasma of a single chroniccarrier donor by methods previously described by Peterson et al., J.Biol. Chem., 256, 6975 (1981). The structural polypeptide (P-25) and thetryptic fragments P-25-1 (residues 1-122) and P-25-2 (residues 123-226)were prepared from this same HBsAg/adw positive donor by preparativepolyacrylamide gel electrophoresis also as described by Peterson et al.,supra. The synthetic peptides shown in FIG. 1 were synthesized by thesolid-phase methods described herein. The polypeptides and trypticfragments were lyophilized, resuspended in culture media as previouslydescribed and were sterilized by gamma irradiation (5000 rads).

B. Immunization

Anti-polypeptide antibodies were produced in rabbits. Polypeptides werecoupled to keyhole limpet hemocyanin (KLH) through the existent or addedcysteine of the polypeptide by usingm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the couplingreagent [See Section VI(c)]. Rabbits were immunized with polypeptide-KLHconjugates according to the following schedule: (1) 200 micrograms ofpolypeptide in complete Freund's adjuvant (CFA) administeredsubcutaneously on day 0; (2) 200 micrograms of polypeptide in incompleteFreund's adjuvant (IFA) on day 14; and (3) 200 micrograms of polypeptidewith 4 milligrams alum administered intraperitoneally on days 21 and 91.The animals were bled 15 weeks after the first injection. Polypeptides1, 72 and 73 were injected without KLH. The above weights of thepolypeptides do not include the weights of the carriers.

To study in vivo antibody production in mice, groups of mice wereimmunized with 4.0 micrograms native HBsAg/ad or 100 micrograms p72 inCFA by intraperitoneal injection. Peptide recipient mice were givenidentical secondary and tertiary immunizations at 2 Week and 4 weekintervals. In vivo priming for the lymph node proliferative assay wasaccomplished by injection of a total of 16.0 micrograms HBsAg in CFA ina volume of 80 microliters into the two hind footpads of the recipientmice.

C. Measurement of anti-HBs.

Anti-HBs antibodies induced by immunization with a synthetic polypeptideor native HBsAg and anti-polypeptide antibodies induced by polypeptideimmunization were measured by two methods. Murine sera were evaluatedfor anti-HBs and anti-polypeptide reactivity in an indirect,immunoglobulin class-specific, radiommunoasay (RIA) utilizingsolid-phase HBsAg (ad or ay subtype) or synthetic peptides, goatanti-mouse IgG, and were developed with ¹²⁵ I-labeled, swine anti-goatIg as described in Milich et al., J. Immunol., 129, 320 (1982).

To analyze rabbit sera for anti-HBs activity, a hemagglutination (HA)system was used. Human type `O`, Rh negative red blood cells were coatedwith HBsAg (ad or ay subtype) by the chromic chloride method asdescribed in Vyas et al., Science, 170, 332 (1970) which is incorporatedherein by reference. The coated cells were added to 0.25 milliliters ofserially diluted test sera in microtiter `V`-bottom plates. All anti-HBsassays were performed in 5-10 percent normal human sera to neutralizeany possible antibodies to contaminating human plasma proteins that maynot have been removed from the HBsAg preparation by the purificationprocedures utilized.

D. Lymph node proliferation assay.

Groups of 5 mice were immunized in the hind footpads with an emulsion ofCFA and 16 micrograms HBsAg (ad or ay subtype). Twelve days laterpopliteal lymph node (PLN) cells were harvested and cultured in vitro toa concentration of 5×10⁵ cells with various challenge antigens. The invitro antigens included native HBsAg [ad (pooled), ay (pooled) or adwfrom a single donor]; polypeptide P25; tryptic fragments P-25-1 andP-25-2; and a synthetic polypeptide of the present invention (P73, P72,P71, P49, P6, P5, P5a, P2, P1).

Draining popliteal lymph node cells were aseptically removed from eachmouse and teased to yield a single cell suspension. The cells werewashed twice with a balanced salt solution (BSS) containingphosphate-buffered saline (pH 7.2). The cells were resuspended inClick's medium containing BSS, L-glutamine, sodium pyruvate,antibiotics, 2-mercaptoethanol, essential and non-essential amino acidsand vitamins. [See Click et al., Cell Immunol., 3, 264 (1972).] Click'smedium, was however modified by the addition of 10 millimolar HEPES(N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid) and gentamycin(10 micrograms per milliliter) and by the substitution of 0.5 percentsyngeneic normal mouse serum for fetal calf serum.

The antigens were tested in culture over a dose range. However, theproliferative responses shown in FIGS. 3 and 4 correspond to thefollowing in vitro doses: native 100 micrograms per milliliter.

Viable lymph node cells (4×10⁵) in 0.1 milliliter of medium were placedin flat-bottom microtiter wells (Falcon 3072, Falcon Plastics, Inc.)with: (a) 0.1 ml. of HBsAg of the ad or ay subtype (2.0 to 0.6micrograms per milliliter), (b) culture medium and ovalbimin (200micrograms per milliliter as a negative control, or (c) purified proteinderivative (PPD-50 micrograms per milliliter) as a positive control.

Cultures were incubated for 5 days at 37 degrees C. in a humidifiedatmosphere containing 5 percent carbon dioxide in air.

On the fourth day, each culture was pulsed with microcurie ³ H-thymidine(³ HTdR) (6.7 Ci/millimole, New England Nuclear, Boston, MA) 16 to 18hours before harvesting. Proliferation was determined by theincorporation of ³ HTdR into DNA. Specific proliferation as astimulation index (SI) that equals the counts per minute (cpm) of thetest antigen divided by the cpm of the media control. It wasdemonstrated previously that the HBsAg-specific proliferation responseof draining PLN cells harvested up to 13 days post-immunization is dueto proliferating T cells [Milich et al., J. Immunol, 130, 1401 (1983)].Therefore, unfractionated PLN cells were used in experiments reportedherein.

VI. Peptide Syntheses and Selection

A. Synthesis of Polypeptides

The polypeptides of this invention were chemically synthesized bysolid-phase methods as described in Merrifield et al., J. Am. Chem.Soc., 85, 2149 (1963) and Houghten et al., Int. J. Peptide ProteinResearch, 16, 311 (1980). The relatively short polypeptides used hereinsubstantially correspond to antigenic determinants of HBsAg.

FIG. 1 shows the 226 amino acid residue sequence of HBsAg. The aminoacid residue sequences of the preferred synthetic polypeptides describedherein are shown in FIGS. 1 and 2. In certain instances, a cysteineresidue was added to the amino-terminus or to the carboxy-terminus ofsome of the polypeptides to assist in coupling to a protein carrier asdescribed below. The compositions of all polypeptides were confirmed byamino acid analysis.

Generally, an immunogen or synthetic polypeptide is made by the steps ofproviding a plurality of amino acids that correspond to the amino acidresidues of an antigenic determinant domain of HBsAg and synthesizingthose amino acids into a polypeptide that has a peptide sequencecorresponding to the peptide sequence of that antigenic determinant. Theproduced synthetic polypeptide can be used to produce a vaccine, usuallyby linking it to a carrier to form a conjugate and then dispersing aneffective amount of the conjugate in a physiologically tolerablediluent.

The polypeptides are preferably synthesized according to theabove-referenced solid phase methods using a cysteine resin. SeeMerrifield et al., supra. The side chains on individual amino acids areprotected as follows: Arg-tosyl, Ser-, Thr-, Glu- and Asp-O-benzyl;Tyr-O-bromobenzyloxy carbamyl; Trp-N-formyl. The N-formyl group on theTrp residues is removed after cleavage of the peptide from the resinsupport by treatment with 1.0 molar ammonium bicarbonate at a peptideconcentration of 1.0 milligram/milliliter for 16 hours at the roomtemperature. Yamashiro et al., J. Org. Chem., 38, 2594-2597 (1973). Theefficiency of coupling at each step can be monitored with ninhydrin orpicric acid and is preferably greater than 99 percent in all cases. SeeGisin, Anal. Chem. Acta, 58, 248-249 (1972) and Kaiser, Anal. Biochem.,34, 595-598 (1980).

Throughout the application, the phrase "immunologically correspondssubstantially" in its various grammatical forms is used herein and inthe claims in relation to polypeptide sequences to mean that thepolypeptide sequence described induces production of antibodies thatbind to the polypeptide and (a) bind to the antigenic determinant ofnative HBsAg for polypeptides 49, 49a, 72 and 72a or (b) induce T cellproliferation for polypeptides 1, 5, 5a, 6 and 71. Thus, the peptides ofthis invention function immunologically as do the corresponding portionsof the HBsAg molecules while also being capable of inducing theproduction of antibodies to themselves.

The term "substantially corresponds" in its various grammatical forms isused herein and in the claims in relation to polypeptide sequences tomean the polypeptide sequence described plus or minus up to three aminoacid residues at either or both of the amino- and carboxy-termini andcontaining only conservative substitutions in particular amino acidresidues along the polypeptide sequence.

The term "conservative substitution" as used above is meant to denotethat one amino acid residue has been replaced by another, biologicallysimilar residue. Examples of conservative substitutions include thesubstitution of one hydrophobic residue such as Ile, Val, Leu or Met foranother, or the substitution of one polar residue for another such asbetween Arg and Lys, between Glu and Asp or between Gln and Asn, and thelike.

In some instances, the replacement of an ionic residue by an oppositelycharged ionic residue such as Asp by Lys has been termed conservative inthe art in that those ionic groups are thought to merely providesolubility assistance. In general, however, since the replacementsdiscussed herein are on relatively short synthetic polypeptide antigens,as compared to a whole protein, replacement of an ionic residue byanother ionic residue of opposite charge is considered herein to be"radical replacement", as are replacements between nonionic and ionicresidues, and bulky residues such as Phe, Tyr or Trp and less bulkyresidues such as Gly, Ile and Val.

The terms "nonionic" and "ionic" residues are used herein in their usualsense to designate those amino acid residues that normally either bearno charge or normally bear a charge, respectively, at physiological pHvalues. Exemplary nonionic residues include Thr and Gln, while exemplaryionic residues include Arg and Asp.

The word "antigen" has been used historically to designate an entitythat is bound by an antibody and to designate the entity that inducesthe production of the antibody. More current usage limits the meaning ofantigen to that entity bound by an antibody, while the word "immunogen"is used for the entity that induces antibody production. In someinstances, the antigen and immunogen are the same entity as where asynthetic polypeptide is utilized to induce production of antibodiesthat bind to the polypeptide. However, the same polypeptide (P49a) canalso be utilized to induce antibodies that bind to a whole protein suchas HBsAg, in which case the polypeptide is both immunogen and antigen,while the HBsAg is an antigen. Where an entity discussed herein is bothimmunogenic and antigenic, it will generally be termed an antigen.

B. Preparation of Polymers

The polypeptides of the present invention can be connected together toform an antigenic polymer (synthetic multimer) comprising a plurality ofthe polypeptide repeating units. Such a polymer has the advantages ofincreased immunological reaction and where different polypeptides areused to make up the polymer, the additional ability to induce antibodiesthat immunoreact with several antigenic determinants of HBsAg.

A polymer (synthetic multimer) can be prepared by synthesizing thepolypeptides as discussed above and by adding cysteine residues at boththe amino- and carboxy-termini to form a "diCys-terminated" polypeptide.Thereafter, in a typical laboratory preparation, 10 milligrams of thediCys polypeptide (containing cysteine residues in un-oxidized form) aredissolved in 250 milliliters of 0.1 molar ammonium bicarbonate buffer.The dissolved diCys-terminated polypeptide is then air oxidized bystirring the resulting solution gently for a period of about 18 hours,or until there is no detectable free mercaptan by the Ellman test. [SeeEllman, Arch. Biochem. Biophys., 82, 70 (1959).]

The polymer (synthetic multimer) so prepared contains a plurality of thepolypeptides of this invention as repeating units. Those polypeptiderepeating units are bonded together by oxidized cysteine residues.

C. Coupling of Polypeptides to Protein Carriers

The synthetic polypeptides were coupled to keyhole limpet hemocyanin(KLH) or tetanus toxoid (TT) by either of the following two methods. Inthe first method, the carrier was activated withm-maleimidobenzoyl-N-hydroxysuccinimide ester and was subsequentlycoupled to the polypeptide through a cysteine residue added to theamino- or carboxy-terminus of the polypeptide, as described in Liu etal., Biochem., 80, 690 (1979). In the second method, the polypeptide wascoupled to the carrier through free amino groups, using a 0.04 percentglutaraldehyde solution as is well known. See, for example, Klipstein etal., J. Inpect. Disc., 147, 318 (1983).

As discussed before, cysteine residues added at the amino- and/orcarboxy-terminii of the synthetic polypeptide have been found to beparticularly useful for forming conjugates via disulfide bonds andMichael addition reaction products, but other methods well known in theart for preparing conjugates can also be used. Exemplary additionalbinding procedures include the use of dialdehydes such as glutaraldehyde(discussed above) and the like, or the use of carbodiimide technology asin the use of a water-soluble carbodiimide, e.g.1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, to form amide links tothe carrier.

Useful carriers are well known in the art and are generally proteinsthemselves. Exemplary of such carriers are keyhole limpet hemocyanin(KLH), edestin, thyroglobulin, albumins such as bovine serum albumin orhuman serum albumin (BSA or HSA, respectively), red blood cells such assheep erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well aspolyamino acids such as poly(D-lysine:D-glutamic acid), and the like.

As is also well known in the art, it is often beneficial to bind thesynthetic polypeptide to its carrier by means of an intermediate,linking group. As noted above, glutaraldehyde is one such linking group.However, when cysteine is used, the intermediate linking group ispreferably an m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS). MBSis typically first added to the carrier by an ester-amide interchangereaction. Thereafter, the above Michael reaction can be followed, or theaddition can be followed by addition of a blocked mercapto group such asthiolacetic acid (CH₃ COSH) across the maleimido-double bond. Aftercleavage of the acyl blocking group, and a disulfide bond is formedbetween the deblocked linking group mercaptan and the mercaptan of theadded cysteine residue of the synthetic polypeptide.

The choice of carrier is more dependent upon the ultimate intended useof the antigen than upon the determinant portion of the antigen, and isbased upon criteria not particularly involved in the present invention.For example, if a vaccine is to be used in animals, a carrier that doesnot generate an untoward reaction in the particular animal should beselected. If a vaccine is to be used in man, then the overridingconcerns involve the lack of immunochemical or other side reaction ofthe carrier and/or the resulting antigen, safety and efficacy--the sameconsiderations that apply to any vaccine intended for human use.

VII. Immunization Procedures

The inocula or vaccines used herein contain an effective amount ofpolypeptide alone, as a polymer of individual polypeptides linkedtogether through oxidized cysteine residues or as a conjugate linked toa carrier. The effective amount of polypeptide per inoculation depends,among other things, on the species of animal inoculated, the body weightof the animal and the chosen inoculation regimen as is well known.Vaccines are typically prepared from the dried solid polypeptide orpolypeptide polymer by suspending the polypeptide or polypeptide polymerin water, saline or adjuvant, or by binding the polypeptide to a carrierand suspending the carrier-bound polypeptide (conjugate) in a similarphysiologically tolerable diluent such as an adjuvant (as previouslydescribed).

These inocula typically contain polypeptide concentrations of about 20micrograms to about 500 milligrams per inoculation. The stated amountsof polypeptide refer to the weight of polypeptide without the weight ofa carrier, when a carrier was used.

The vaccines also contained a physiologically tolerable (acceptable)diluent such as water, phosphate-buffered saline or saline, and furthertypically include an adjuvant. Adjuvants such as complete Freund'sadjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum arematerials well known in the art, and are available commercially fromseveral sources.

Vaccine stock solutions were prepared with CFA, IFA or alum as follows:An amount of the synthetic polypeptide, polymeric polypeptide orconjugate sufficient to provide the desired amount of polypeptide perinoculation was dissolved in phosphate-buffered saline (PBS) at a pHvalue of 7.2. Equal volumes of CFA, IFA or alum were then mixed with thepolypeptide solution to provide a vaccine containing polypeptide, waterand adjuvant in which the water-to-oil ratio was about 1:1. The mixturewas thereafter homogenized to provide the vaccine stock solution.

Rabbits were injected subcutaneously and intraperitoneally, aspreviously described, with a vaccine comprising 200 to 400 micrograms ofa polypeptide conjugate emulsified in complete Freund's adjuvant (CFA),incomplete Freund's adjuvant (IFA) or alum (5 milligrams per milliliterin each instance) on days 0, 14 and 21, respectively. Each inoculation(immunization) consisted of four injections of the inoculum. Mice wereimmunized in a similar way using one tenth of the above dose perinjection.

Animals were bled 7 and 14 days after the last injection. In some cases,the animals received booster injections in alum, and were bledthereafter as necessary. Control pre-immune serum was obtained from eachanimal by bleeding just before the initial immunization.

Inoculum stock solutions can also be prepared with keyhole limpethemocyanin (KLH), KLH in IFA (incomplete Freund's adjuvant), KLH-alumabsorbed, KLH-alum absorbed-pertussis, edestin, thyroglobulin, tetanustoxoid, tetanus toxoid in IFA, cholera toxoid and cholera toxoid in IFA.

Upon injection or other introduction of the antigen or vaccine into thehost, the immune system of the host responds by producing large amountsof antibody to the antigen. Since the specific antigenic determinant ofthe manufactured antigen, i.e., the antigen formed from the syntheticpolypeptide and the carrier immunologically corresponds substantially tothe determinant of the natural antigen of interest, the host becomesimmune to the natural antigen. In the case where the invention is usedas a vaccine, this is the desired result.

VIII. Delayed-Type Hypersensitivity (Skin Reaction Test)

The previously described diagnostic systems and assays are based on invitro assays. Although particular steps of the assays can be carried outin vivo, the actual immune response is measured in tissue culture. Thepresent invention, however, can also be applied to diagnostic systemsinvolving the in vivo measurement of T cell responses. One example ofsuch a system is a delayed-type hypersensitivity (DTH) reaction or whatis more commonly known as a skin reaction test.

A DTH reaction can only occur in an individual previously exposed(sensitized) to a given antigen. The first exposure of an individual tothe antigen produces no visible change, but the immune status of theindividual is altered in that hypersensitivity to renewed exposure tothat antigen results. Thus, upon intradermal or subcutaneous injectionof the antigen (preferably in a buffered saline solution) acharacteristic skin lesion develops at the injection site--a lesion thatwould not develop after a first antigen exposure. Because the responseto the second (or challenge) antigen inoculum is typically delayed by 24to 48 hours, the reaction is referred to as delayed-typehypersensitivity.

In humans, exposure to a sensitizing antigen takes place upon contactwith the microorganism responsible for the disease (e.g., tuberculinfrom Mycobacterium tuberculosis, typhoidin from Salmonella typhi andabortin from Brucella abortus), and sensitization occurs as a result ofa chronic infection. In animals, sensitization can be achieved byinoculation of an antigen emulsified in water, saline or an adjuvant.

In both humans and animals, hypersensitivity is tested in vivo by theinjection of the antigen dissolved in a physiologically tolerablediluent such as saline solution into the skin (either intradermally orsubcutaneously). DTH is usually a more sensitive diagnostic assay thanthe determination or measurement of the amount of antibody produced toan antigen. For example, only minute amounts of protein (a few hundredmicrograms) are necessary for DTH sensitization of a mouse, while a muchlarger dose is needed to induce antibody production.

Since the polypeptides of the present invention (in particular,polypeptide P71) stimulate the proliferation of human and murine T cellsfollowing immunization (sensitization) with active HBsAg or withpolypeptides 49, 49a, 72 and 72a, a skin reaction test was developedusing one or more of the present synthetic polypeptides as a challengeantigen.

Selected murine strains are immunized with native HBsAg emulsified on anadjuvant such as complete Freund's adjuvant (CFA) by intradermalinjection in the flank. In experimental situations, DTH sensitizationusually occurs only when the sensitizing antigen is administered inadjuvant, preferably the complete type that includes bacilli oftuberculosis.

Seven days after immunization, the mice are challenged by intradermalinoculation in the ear or in the footpad with a predetermined amount ofan antigen including (a) native HBsAg or (b) one or more of the presentsynthetic polypeptides in a known volume of phosphate-buffered saline(PBS). Control mice are inoculated intradermally with the same volume ofPBS not including the antigen. Additional controls include miceimmunized with only CFA.

Thickening of the tissue at the antigen-injection site relative to thecontrol sites is evidence of a DTH reaction. Thus, the thickness of theears and footpads is measured before challenge with the antigen and at4, 24 and 48 hours after challenge.

Results demonstrate that the synthetic polypeptides of the presentinvention (in particular, P71) may be useful in an in vivo murinediagnostic system for the presence of a cell mediated immune response toHBsAg.

After the safety and effectiveness of the above polypeptides are shownin animal studies, the polypeptides can be used as challenge antigens inhuman skin reaction tests for recipients of HBsAg vaccines. Thepolypeptides are synthesized as previously described, purified by highpressure liquid chromatography (HPLC) techniques, sterilized andpyrogen-tested.

Since the T cell proliferative responses of human HBsAg vaccinerecipients can be quite variable relative to polypeptide specificity,vaccine recipients and individuals serving as unvaccinated controls arechallenged with a series of polypeptides. The kinetics and optimalantigen dose can be determined in the vaccine recipient group using theresults from the animal studies as a guideline.

HBV acute and chronically infected individuals can also be studied forHBsAg-specific T cell sensitization using synthetic polypeptides asantigens for a skin reaction test.

In each instance, the challenge antigen is administered by intradermalinjection of the particular polypeptide in a physiologically acceptablesolution (about 1 milliliter) into the volar surface of the forearm. Useof a 25- or 27-gauge needle usually assures intradermal rather thansubcutaneous administration of the antigen. Subcutaneous injection canlead to dilution of the antigen in tissues and can produce afalse-negative test. The injection sites are then observed for erythema(skin reddening) and induration (swelling) at 4, 24 and 48 hourspost-challenge.

The foregoing is intended as illustrative of the present invention butis not limiting. Numerous variations and modifications can be madewithout departing from the spirit and scope of the novel concepts of theinvention. It should be understood that no limitation with respect tothe specific compositions and uses described herein is intended orshould be inferred.

What is claimed is:
 1. A vaccine against infection by hepatitis B viruscomprising:(a) an effective amount of a synthetic polypeptide having anamino acid residue sequence taken from left to right and in thedirection from amino-terminus to carboxy-terminus, and represented bythe formula: ThrLysProSerAspGlyAsnCysThrCysIleProIleProSer;(b) aneffective amount of at least one synthetic polypeptide having an aminoacid residue sequence taken from left to right and in the direction fromamino-terminus to carboxy-terminus selected from the group consistingof: PheProGlySerSerThrThrSerThrGlyProCysArgThrCysMetThrThrAlaGlnGlyThrSerMetTyrProSerCys;MetThrThrAlaGlnGlyThrSerMetTyrProSerCys;IleProGlySerThrThrThrSerThrGlyProCysLysThrCysThrThrProAlaGlnGlyAsnSerMetPheProSerCys;ThrThrProAlaGlnGlyAsnSerMetPheProSerCys; andCysProLeuIleProGlySerThrThrThrSerThrGlyProCysLysThrCysThrThrProAlaGlnGlyAsnSerMet PheProSerCys; and(c) aphysiologically tolerable diluent, said vaccine when introduced into ahost, being capable of inducing the production of antibodies and theproliferation of thymus-derived cells in the host, said antibodiesimmunoreacting with said hepatitis B virus, and said vaccine protectingthe host from hepatitis B viral infection.
 2. The vaccine according toclaim 1 wherein said physiologically tolerable diluent is a member ofthe group consisting of water, saline and an adjuvant.
 3. The vaccineaccording to claim 1 wherein said synthetic polypeptides are bound to acarrier.
 4. The vaccine according to claim 1 wherein said carrier isselected from the group consisting of keyhole limpet hemocyanin, keyholelimpet hemocyanin in incomplete Freund's adjuvant, alum, keyhole limpethemocyanin-alum absorbed, keyhole limpet hemocyanin-alumabsorbed-pertussis, edestin, thyroglobulin, tetanus toxoid, tetanustoxoid in incomplete Freund's adjuvant, cholera toxoid and choleratoxoid in incomplete Freund's adjuvant.
 5. A vaccine against infectionby hepatitis B virus comprising an effective amount of a syntheticpolypeptide having an amino acid residue sequence shorter than that ofhepatitis B virus surface antigen that immunologically correspondssubstantially to limited portions of an amino acid residue sequence ofthe hepatitis B virus surface antigen, said limited portions being (a)from about positions 140 to 154 and (b) from about positions 110 to 137from the amino-terminus thereof, and a physiologically tolerablediluent, said vaccine when introduced into a host, being capable ofinducing the production of antibodies and the proliferation ofthymus-derived cells in the host, said antibodies immunoreacting withsaid hepatitis B virus and said vaccine protecting the host fromhepatitis B viral infection.
 6. The vaccine according to claim 5 whereinthe synthetic polypeptide includes the sequences of amino acid residuestaken from left to right and in the direction from amino-terminus tocarboxy-terminus, and represented by the formulae:ThrLysProSerAspGlyAsnCysThrCysIleProIleProSer; andPhe(Ile)ProGlySerSer(Thr)ThrThrSerThrGlyProCysArg(Lys)ThrCysMet(Thr)ThrThr(Pro)AlaGlnGlyThr(Asn)SerMetTyr(Phe)ProSerCyswherein each amino acid residue inparentheses is an alternative to the immediately preceding amino acidresidue.
 7. A vaccine against infection by hepatitis B virus comprisingan effective amount of a synthetic polypeptide having an amino acidresidue sequence shorter than that of hepatitis B virus surface antigenthat immunologically corresponds substantially to a limited portion ofan amino acid residue sequence of a natural pathogen-related proteinencoded by a hepatitis B virus surface antigen, said limited portionbeing from about positions 110 to 154 from the amino-terminus thereof,and a physiologically tolerable diluent, said vaccine when introducedinto a host, being capable of inducing the production of antibodies andthe proliferation of thymus-derived cells in the host, said antibodiesimmunoreacting with said hepatitis B virus and said vaccine protectingthe host from hepatitis B viral infection.
 8. The vaccine according toclaim 7 wherein the synthetic polypeptide includes the sequence of aminoacid residues taken from left to right and in the direction fromamino-terminus to carboxy-terminus, and represented by the formula:Phe(Ile)ProGlySerSer(Thr)ThrThrSerThrGlyProCysArg(Lys)ThrCysMet(Thr)ThrThr(Pro)AlaGlnGlyThr(Asn)SerMetTyr(Phe)ProSerCysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIleProSerwherein each amino acidresidue in parentheses is an alternative to the immediately precedingamino acid residue.
 9. A vaccine against infection by hepatitis B viruscomprising an effective amount of a synthetic multimer in aphysiologically tolerable diluent, said synthetic multimer comprising aplurality of polypeptide repeating units including(a) an amino acidresidue sequence taken from left to right and in the direction fromamino-terminus to carboxy-terminus, and represented by the formula:ThrLysProSerAspGlyAsnCysThrCysIleProIleProSer; and(b) at least one aminoacid residue sequence taken from left to right and in the direction fromamino-terminus to carboxy-terminus selected from the group consistingof: PheProGlySerSerThrThrSerThrGlyProCysArgThrCysMetThrThrAlaGlnGlyThrSerMetTyrProSerCys;MetThrThrAlaGlnGlyThrSerMetTyrProSerCys;IleProGlySerThrThrThrSerThrGlyProCysLysThrCysThrThrProAlaGlnGlyAsnSerMetPheProSerCys;ThrThrProAlaGlnGlyAsnSerMetPheProSerCys; andCysProLeuIleProGlySerThrThrThrSerThrGlyProCysLysThrCysThrThrProAlaGlnGlyAsnSerMet PheProSerCys;wherein at leasttwo Cys residues are present and said synthetic multimer contains atleast one intramolecular cystine disulfide bond formed from at least twoof the Cys residues present, said vaccine when introduced into a host,being capable of inducing the production of antibodies and theproliferation of thymus-derived cells in the host, said antibodiesimmunoreacting with said hepatitis B virus, and said vaccine protectingthe host from hepatitis B viral infection.
 10. The vaccine according toclaim 9 wherein said physiologically tolerable diluent is a member ofthe group consisting of water, saline and an adjuvant.
 11. The vaccineaccording to claim 9 wherein said synthetic multimer is bound to acarrier.
 12. The vaccine according to claim 9 wherein said carrier isselected from the group consisting of keyhole limpet hemocyanin, keyholelimpet hemocyanin in incomplete Freund's adjuvant, alum, keyhole limpethemocyanin-alum absorbed, keyhole limpet hemocyanin-alumabsorbed-pertussis, edestin, thyroglobulin, tetanus toxoid, tetanustoxoid in incomplete Freund's adjuvant, cholera toxoid and choleratoxoid in incomplete Freund's adjuvant.
 13. The vaccine according toclaim 9 wherein the intramolecular cystine disulfide bond of saidsynthetic multimer is an intrapolypeptide disulfide bond.
 14. Thevaccine according to claim 9 wherein the polypeptide repeating units ofsaid synthetic multimer are bonded together head-to-tail through anamide bond formed between the amine group of the amino-terminal residueof a first polypeptide repeating unit and the carboxyl group of thecarboxy-terminal residue of a second polypeptide repeating unit.
 15. Thevaccine according to claim 14 wherein said synthetic multimer containsabout two to about three of said polypeptide repeating units.
 16. Thevaccine according to claim 9 wherein the intramolecular cystinedisulfide bond of said synthetic multimer is an interpolypeptidedisulfide bond.
 17. The vaccine according to claim 16 wherein thepolypeptide repeating units of said synthetic multimer are bondedtogether by said interpolypeptide cystine disulfide bond formed betweenthe Cys residues of said polypeptide.
 18. A vaccine against infection byhepatitis B virus comprising a physiologically tolerable diluent havingdispersed therein (i) an effective amount of a synthetic polypeptidehaving an amino acid residue sequence that immunologically correspondssubstantially to a portion of an amino acid residue sequence of thehepatitis B virus surface antigen from about positions 110 to 137 fromthe amino-terminus thereof and (ii) an effective amount of a syntheticpolypeptide having an amino acid residue sequence taken from left toright and in the direction from amino-teminus to carboxy-teminus, andrepresented by the formula: ThrLysProSerAspGlyAsnCysThrCysIleProIleProSer;said vaccine, when introduced into a host, beingcapable of inducing the production of antibodies and the proliferationof thymus-derived cells in the host, said antibodies immunoreacting withsaid hepatitis B virus and said vaccine protecting the host fromhepatitis B viral infection.
 19. A vaccine against infection byhepatitis B virus comprising a physiologically tolerable diluent havingdispersed therein an effective amount of a synthetic polypeptide havingan amino acid residue sequence taken from left to right and in thedirection from amino-terminus to carboxy-terminus, and represented bythe formula: Phe(Ile)ProGlySerSer(Thr)ThrThrSerThrGlyProCysArg(Lys)ThrCysMet(Thr)ThrThr(Pro)AlaGlnGlyThr(Asn)SerMetTyr(Phe)ProSerCysCysCysThrLysProSerAspGlyAsn CysThrCysIleProIleProSer,whereineach amino acid residue in parentheses is an alternative to theimmediately preceding amino acid residue, said vaccine, when introducedinto a host, being capable of inducing the production of antibodies andthe proliferation of thymus-derived cells in the host, said antibodiesimmunoreacting with said hepatitis B virus and said vaccine protectingthe host from hepatitis B viral infection.